ABSTRACT Most human cancers have chromosomal structural changes, and double-strand DNA breaks (DSBs) are the major cause. Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing DSBs, and NHEJ is sufficiently flexible that it can join any pair of DNA ends, regardless of their structure. The flexibility of NHEJ is essential because natural causes of DSBs (e.g., ionizing radiation, reactive oxygen species, failed nuclear enzyme reactions) generate DNA ends with diverse molecular configurations. Hence, that flexibility is well suited for the task, but it has the negative impact of causing DNA sequence alterations at nearly all repair sites. NHEJ represents one of the most sophisticated protein:DNA interaction pathways because transiently there is no covalent connection between the two DNA ends. Although we know most of the proteins that participate in NHEJ and know, in broad terms, how they function, we do not have a clear picture of their spatial and temporal interactions, or how some components are required for some NHEJ events but not others, depending on the DNA end molecular configuration. With a clearer mechanistic and structural picture of human NHEJ, we will be in a position to develop small molecule inhibitors that may be useful for treating many cancers (e.g., chemotherapeutically or as a radiation sensitizer). Aim 1 describes our innovative steps toward elucidating the structure of the Artemis:DNA-PKcs:Ku complex. Though a few individual proteins in the complex have known structures (e.g., Ku and portions of DNA-PKcs), we want to understand how different assemblies of the NHEJ proteins, such as Ku, DNA-PKcs, and Artemis, are needed to repair the various DNA end molecular configurations that arise in the cell. Aims 2A and 2B describe our ability to directly follow the chemical steps of NHEJ using purified proteins and our ability to dissect which steps are critical for each type of DNA end configuration. Aim 2C and 2D determine how wrapping the DNA duplex around histone octamers (mononucleosomes) affects the NHEJ joining mechanism and chemistry of the steps. Aim 3 examines the noncovalent approximation (synapsis) of the two DNA ends during NHEJ, which is optimally studied using sm-FRET. Aim 3A determines the extent to which the synapsis step (approximiation of the two DNA ends) determines the overall rate of NHEJ. Aim 3B uses sm-FRET to test whether nucleosomal DNA can be synapsed by NHEJ proteins. Aim 4 describes our innovative development of small molecule inhibitors of Artemis, which is an essential nuclease for resolving DNA repair intermediates due to damage from ionizing radiation or topoisomerase II inhibitors (both of which are used therapeutically). A high throughput screen of 433,000 compounds has identified 20 compounds that merit further study, and a subset satisfy a highly stringent human cellular bioselectivity test in which a specific half of the reaction products are blocked without affecting the other half or DNA metabolism.